Project Summary/Abstract Macroautophagy is a vital process in which cells degrade malformed proteins and damaged macromolecules and organelles using the lysosomal machinery. Autophagy initiates with formation of an autophagophore in the vicinity of the endoplasmic reticulum (ER), followed by its expansion, maturation, and closure to form an autophagosome. In the final step, the autophagosome fuses with a lysosome to initiate the degradation and recycling of the engulfed cargoes. Although the later stages of autophagy have been studied thoroughly, little is known about the early stages of phagophore biogenesis. Recently our group discovered that ATG2, a protein essential for autophagy, contains an elongated cavity that can solubilize and transfer lipids between membranes in vitro, and that it mediates lipid transfer from the ER to the nascent phagophore. Mutational impairment of ATG2-mediated lipid transfer attenuates autophagy in vivo, suggesting this function is critical to autophagophore growth. The long-term goal of this application is to elucidate the underlying molecular events that drive phagophore membrane biogenesis by better understanding the role of lipid transport mediated by ATG2 and any protein partners in autophagy. These protein partners include an integral membrane complex comprising TMEM41B and VMP1, as well as other early autophagy proteins. In Aim 1, I will biochemically study the mechanism of several key early-autophagy proteins at sites of autophagophore initiation to better understand the machinery required to support membrane expansion. In Aim 2, I will use structural and biochemical approaches to inspect the specific function of an ER-localized integral membrane complex, TMEM41B and VMP1 proteins, which has been shown to be essential in autophagophore biogenesis.